Repellency of Anethole- and Estragole-Type Fennel Essential Oils Against Stored Grain Pests: the Different Twins

Bulletin of Insectology 69 (1): 149-157, 2016 ISSN 1721-8861 Repellency of anethole- and estragole-type fennel essential oils against stored grain pests: the different twins 1 2 3 1 2 Stefano BEDINI , Hind Houria BOUGHERRA , Guido FLAMINI , Francesca COSCI , Kamel BELHAMEL , 3 1 Roberta ASCRIZZI , Barbara CONTI 1Department of Agriculture, Food and Environment, University of Pisa, Italy 2Lab. of Organic Materials, Department of Process Engineering, Faculty of Technology, University of Bejaia, Algeria 3Department of Pharmacy, University of Pisa, Italy Abstract Aromatic plants essential oils (EOs) are promising alternatives to chemical insecticides and insect repellent for the post-harvest protection of crops. Fennel (Foeniculum vulgare Mill.) is a highly aromatic plant, cultivated worldwide of which several chemotypes can be distinguished on the basis of the relative content of its main compounds (E)-anethole and estragole. Fennel is well known for its pharmacological, antioxidant antimicrobial and acaricidal activities, and several studies showed its effectiveness as insecticidal and repellent against insects. In this study the repellency of the EOs extracted from two chemotypes, anethole- and estragole-type of F. vulgare fruits, against Rhyzopertha dominica (F.), Sitophilus zeamais Motschulsky and Tribolium confusum Jaqcquelin du Val, three of the major worldwide post-harvest grains insects was assessed by in vitro bioassays. Along with the EOs, we also tested the repellency of their major chemical components (E)-anethole, estragole, limonene and, fenchone and we evaluated the co-repellency effect of (E)-anethole and estragole. Finally, the repellence of the fennel EOs in the presence of maize was tested by a two-choice pitfall bioassay. (E)-anethole and estragole content in the anethole-type was 78.4 and 8.0%, respectively while in the estragole-type fennel EO anethole and estragole content was 0.9 and 85.5%, respectively. RD50 values showed that the estragole-type EO was the most effective repellent against the three insect species with values of 0.007, 0.051 and 1.124 mg cm-2 for R. dominica, T. confusum and S. zeamais, respectively. Consistently, relative median potency analysis showed that estragole was significantly more repellent to the three pest insect species than (E)-anethole. Interestingly, in the EOs, (E)-anethole and estragole showed a synergistic co-repellent effect. The strongest synergy was observed against R. dominica (CRC = 634.51). The repellency of fennel EOs also in the presence of maize was confirmed by a two-choice bioassay. Also in this case, the most overall effective EO resulted to be the estragole-type. The results highlight the importance of a chemical standardization based on the bioactivity of the fennel EOs and indicate the estragole-type fennel EO as suitable for the development of eco-friendly repellents for the post-harvest protection of grain crops. Key words: post-harvest pest, anethole; estragole, insect repellency, synergy, fennel. Introduction the potential variability of the EOs effectiveness. Fennel, Foeniculum vulgare Mill. (Apiaceae), is an Insects are major pests of stored food causing losses es- aromatic plant indigenous of the Mediterranean region timated around 20% of the annual world crop produc- well known for its pharmacological properties as carmi- tion (Sallam, 1999). Essential oils (EOs) of aromatic native, digestive, lactogogue and diuretetic (Manzoor et plants are effective natural products as contact and fu- al., 2012). The fennel, is largely distributed, especially migant insecticides and as repellents against stored food in dry soils, near the sea coast and on river banks, both pests (Isman, 2000; Nerio et al., 2009; Conti et al., as wild and cultivated plant (Napoli et al., 2010). The 2011; Athanassiou et al., 2013; Bougherra et al., 2015). species shows a large morphological and chemical di- Moreover, due to their high biodegradability and low versity. Some of the fennel genotypes that have been mammalian toxicity, EOs are regarded as very promis- entered in the European Pharmacopeia, are impossible ing tools for the formulation of rational low-toxic, eco- to be discriminated in relation to only morphological friendly food preservative (Isman, 2006). However, a data. However, several different chemotypes have been major difficulty for the implementation of aromatic identified on the basis of the EOs relative presence of plant EOs is that their bioactivity varies considerably the main compounds, the two enantiomers (E)-anethole depending to their chemical composition. EOs composi- and estragole (Krüger and Hammer, 1999). tion is reported to vary depending on a number of fac- Fennel EO has been already shown to have acaricidal tors such plant genetic structure (Telci et al., 2009), (Lee, 2004), antifungal (Singh et al., 2006), as well as phenological stage and site of origin of the plants antibacterial activity (Ruberto et al., 2000) and, re- (Shaaya and Kostyukovysky, 2006). Since the biologi- cently, its effectiveness as insecticidal and repellent cal activities of EOs depend on their chemical composi- against insects has been evaluated as well (Bertoli et al., tion, the chemical characterization coupled with the as- 2012). However, to our knowledge, no information is sessment of their biological properties is essential to available about the fennel EO effectiveness in relation standardize the chemical parameters that ensure the EOs to the chemical composition of its chemotypes. biological effects. Such standardization that has already The aim of the work was to assess the repellent activity been successfully introduced for the Australian Tea tree of different fennel EO chemotypes and of their main oil (Callander and James, 2012), could strongly reduce compounds against three of the major grain pests: the lesser grain borer Rhyzopertha dominica (F.) (Bostrichi- Chemicals dae), the maize weevil Sitophilus zeamais Motschulsky (E)-anethole, estragole, limonene and, fenchone were (Dryophthoridae), and the confused flour beetle Tri- purchased from Sigma-Aldrich (Milano, Italy). Chemi- bolium confusum Jaqcquelin du Val (Tenebrionidae) in cals were of analytical standard grade. order to detect the relevant chemical parameters useful for a bioactivity based standardization of the fennel EOs. Insect cultures and rearing conditions R. dominica, S. zeamais and T. confusum were reared at the Department of Agriculture, Food and Environ- Materials and methods ment of the University of Pisa, since 2000. Insects were reared at room temperature (20-24 °C), 45-65% R.H., in Plant material the dark, in (20 × 27 × 11 cm) plastic boxes containing Estragole-type F. vulgare fruits were manually broken corn and wheat and covered by a nylon net al- harvested, at full ripeness, in Kabylia (Algeria) in the lowing air exchange. Insects homogeneous in age for summer 2012, dried in the shade, at room temperature the bioassays were obtained by removing the adults pre- (20-25 °C) until constant weight and stored in sent in the rearing boxes by sieving the grain and col- polyethylene bags in the dark at 4 °C. Seeds were then lecting the newly emerged insects (S. zeamais, 0-3 days transported in insulated polystyrene boxes on ice to the old; R. dominica and T. confusum, 0-1 day) the follow- laboratory of the University of Pisa for the EO ing day. extraction. Anethole-type F. vulgare fruits, were purchased from Repellence bioassays Biochimica srl (Arezzo, Italy). Area preference bioassays The repellence of the anethole- and estragole-type Essential oil extraction and GC-MS analyses fennel EOs and of their main chemical compounds (E)- Fennel fruits were coarsely grounded in a mortar with anethole, estragole, limonene and, fenchone was as- a pestle and then subjected to hydro-distillation in a sessed by the area preference method described by Ta- modified Clevenger-type apparatus for 3 h (Guenter, pondjou et al. (2005). In detail, half filter paper disks (8 1949). The resulting essential oil was dried over anhy- cm ⌀) were treated with 500 µL of F. vulgare EO or drous sodium sulphate and stored in a glass vial at 4 °C pure chemical compounds as ethanolic solutions. Con- until use. trol half filter paper disks were treated with 500 µL of Gas chromatography (GC) analyses were carried out ethanol. Ethanol of the treated and control filter paper with an Hewlett Packard -5890 Series II instrument disks were evaporated under a fume hood. Each Petri equipped with HP-WAX and HP-5 capillary columns dish’s bottom (8 cm ⌀) was covered with half filter pa- (30 m × 0.25 mm, 0.25 µm film thickness), working with per treated with the EO or the chemical solution, while the following temperature program: 60 °C for 10 min, the other half, was covered with a control half filter pa- ramp of 3 °C min-1 up to 220 °C; injector and detector per disk. Repellency bioassays were performed with temperatures 250 °C; carrier gas helium (2 ml min-1); both F. vulgare EOs at doses ranging from 0.005 to detector dual FID; split ratio 1:30; injection of 0.5 µl 0.385 mg cm-2. Chemicals were tested at doses ranging (10% hexane solution). Components identification was from 0.01 to 0.74 mg cm-2. Twenty unsexed adults were carried out, for both columns, by comparing their reten- introduced in each Petri dish, and the lid was sealed tion times with those of pure authentic samples and by with Parafilm®. The Petri dishes were maintained in means of their linear retention index (LRI), relative to climatic chamber at 25 ± 1 °C, 65 ± 5% R.H., in the the series of n-hydrocarbons. Gas chromatography- dark, covered by black plastic pots. Five replicates were electron impact mass spectroscopy (GC-EIMS) analyses performed for each assay, and insects were used only were performed with a Varian CP-3800 gas chromato- once. The number of insects on the two half of the Petri graph, equipped with a HP-5 capillary column (30 m × dish was recorded after 24 h from the beginning of the 0.25 mm; coating thickness 0.25 µm) and a Varian Sat- test.

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